Barriers to inhaled gene therapy of obstructive lung diseases: A review

Abstract

Knowledge of genetic origins of obstructive lung diseases has made inhaled gene therapy an attractive alternative to the current standards of care that are limited to managing disease symptoms. Initial lung gene therapy clinical trials occurred in the early 1990s following the discovery of the genetic defect responsible for cystic fibrosis (CF), a monogenic disorder. However, despite over two decades of intensive effort, gene therapy has yet to help patients with CF or any other obstructive lung disease. The slow progress is due in part to poor understanding of the biological barriers to inhaled gene therapy. Encouragingly, clinical trials have shown that inhaled gene therapy with various viral vectors and non-viral gene vectors is well tolerated by patients, and continued research has provided valuable lessons and resources that may lead to future success of this therapeutic strategy. In this review, we first introduce representative obstructive lung diseases and examine limitations of currently available therapeutic options. We then review key components for successful execution of inhaled gene therapy, including gene delivery systems, primary physiological barriers and strategies to overcome them, and advances in preclinical disease models with which the most promising systems may be identified for human clinical trials.

Keywords: Biological barrier; Gene delivery vector; Preclinical model; Respiratory gene therapy.

Figure 1. Viral vectors for inhaled gene therapy

(A, B) A recombinant AAV2/5 (AAV2 rep gene, AAV5 cap gene) delivered by intratracheal instillation demonstrates long-lasting gene expression (up to 15 months) in both conducting airways and alveoli of mice. (A) Immunohistological staining of β-galactosidase (β-gal) expression in alveoli and conducting airways 1 month post-administration. (B) Bioluminescence imaging of firefly luciferase expression in the lung and nose of mice at 1, 3, 6, 12, and 15 months post-administration. Reprinted from [105] with permission of Mary Ann Liebert, Inc. (C, D) A simian immuno-deficiency virus pseudotyped with the respiratory pathogen Sendai virus (F/HN-SIV) demonstrates sustained transgene expression in the nose and lungs of mice after intranasal administration, lasting 22 months postadministration. (C) Fluorescent microscopy images of GFP expression mediated by F/HN-SIV in mouse lungs. (D) Bioluminescence imaging of firefly luciferase expression in the mouse lungs and noses at 2 and 22 months post-administration. Reprinted from [131] with permission of the American Thoracic Society. Copyright © 2016 American Thoracic Society.

Figure 2. Non-viral gene vectors for inhaled gene therapy

(A, B) Localization of gene expression in sheep lungs treated with GL67A carrying human CFTR (hCFTR)-expressing plasmid DNA. (A) Dual labeling of two hCFTR epitopes (G449, Texas red; MATG1061, FITC) of lung sections. Arrows indicate epithelial cells that are positive with both antibodies. (B) Dual labeling of cytokeratin and G449 (anti-cytokeratin antibody, red; G449, FITC). Reprinted from [14] with permission of Macmillan Publishers Ltd. (C, D) Localization of gene expression in the (C) medium (20× maginification) and (D) small airways (40× magnification) in the mouse lungs. Lungs harvested from animals that received 100 µg of β-gal expressing plasmid DNA compacted with CK₃₀ PEG_10k were fixed, sectioned, and immunohistochemically stained for the bacterial β-galactosidase protein 2 days after intratracheal administration. Reprinted from [181] with permission of Macmillan Publishers Ltd.

Figure 3. Primary physiological barriers to inhaled gene therapy

(A) Mucus: A confocal image showing 89-nm polystyrene nanoparticles (PS NP) trapped via adhesive interactions within CF mucus. Reprinted from [207] with permission from Elsevier. (B) The periciliary layer (PCL): Adenovirus (AdV, blue arrows) is excluded from the PCL while adeno-associated virus (AAV, red arrows) penetrates into PCL and reaches underlying epithelium. Reprinted from [240] by permission from Macmillan Publishers Ltd. (C) Alveolar macrophages: an SEM image showing aerosolized 110 nm silver nanoparticles (Ag NP) accumulating in macrophages collected from broncheoalveolar lavage fluid (BALF) after being administered to rats. Reprinted from [260] by permission from Oxford University Press. (D) Epithelial cell tight junctions: A confocal image (top: xy view, bottom: xz view) showing AdV restricted to the apical side of human airway epithelium due to the presence of tight junctions which prevents the access to receptors required for cell entry. Reprinted from [272] with permission of the American Thoracic Society. Copyright © 2016 American Thoracic Society.

Figure 4. Modification of gene vectors to overcome physiological barriers

Representative trajectories of (A) conventional DNA nanoparticles (DNA-CP) and (B) mucus-penetrating DNA nanoparticles (DNA-MPP) based on biodegradable PBAE polymers in freshly expectorated CF mucus. Representative images of gene vector distribution in large airways following intratracheal administration of (C) DNA-CP and (D) DNA-MPP. Reprinted from [284] with permission from PNAS. Copyright © 2016 National Academy of Sciences, USA. (E, F) In vivo and (G, H) in vitro transduction of AAV6 and a mutant variants, AAV6.2, engineered with a single amino acid substitution in the heparin binding domain. Comparison of intratracheally administered (E) AAV6 and (F) AAV6.2 showing a stronger β-gal transgene expression in the mouse lung with AAV6.2. Comparison of (G) AAV6- and (H) AAV6.2–mediated GFP expression in ALI cultures of primary HAE. AAV6.2 treated cultures show stronger GFP expression as well as transduction of both ciliated and nonciliated cells (H, inset). Reprinted from [109] by permission from Macmillan Publishers Ltd.

Figure 5. Modulating physiological barriers to inhaled gene therapy

(A, B) N-acetyl cysteine (NAC) treatment increases mucus mesh spacing, thereby facilitating gene vector penetration through airway mucus. Reprinted from [309] with permission from Nanomedicine as agreed by Future Medicine Ltd. (C, D) Reducing the osmotic pressure (OP) of mucus that can be achieved inhaled hypertonic saline rehydrates and restores the collapsed PCL. Reprinted from [238] with permission from AAAS. (E, F) Sodium caprate (C10) disrupts tight junctions in epithelial layer allowing gene vectors to access the basolateral compartment where specific receptors required for viral gene transduction are present. Reprinted from [272] with permission of the American Thoracic Society. Copyright © 2016 American Thoracic Society. The American Journal of Respiratory Cell and Molecular Biology is an official journal of the American Thoracic Society.

Figure 6. Animal models of obstructive lung diseases

Airway obstruction develops in CF pig due to highly viscoelastic mucus (A; white arrow) in the airway where mucus cytology further revealed presence of neutrophils, macrophages, and bacteria (B). Reproduced from [351] with permission from AAAS. Airspace in (C) healthy guinea pigs and (D) guinea pigs exposed to cigarette smoke (CS) for 6 months that show airway enlargement characteristic of emphysema in COPD. Reproduced from [374] with permission from APS. (E) Mucous cell hyperplasia, subepithelial fibrosis, and smooth muscle hypertrophy in ovalbumin (OVA)-induced allergic asthma mouse model. Reproduced from [77] with permission from Elsevier.